Papillomaviruses are ubiquitous, small, non-enveloped viruses with a double-stranded DNA genome of about 8 kbp. Papillomaviruses infect squamous basal epithelial cells through wounds or abrasions and encode about 10 proteins involved in viral DNA replication and structure, and in cell transformation (Burd E M., Clin. Microbiol. Rev. (2003), 16(1):1-17; and Psyrri A, DiMaio D., Nat. Clin. Pract. Oncol. (2008), 5(1):24-31). Duplication of the viral genome absolutely requires host-cell DNA replication components which would otherwise be relatively inactive at this stage of host-cell differentiation but are activated by virally encoded E6 and E7 proteins. The E6 and E7 proteins interact with various host-cell proteins, of which TP53 and RB1, respectively, have been determined to be the functional targets effecting reactivation of cell cycle progression and viral DNA replication. Overall, the process is not cytolytic, with viral DNA evident throughout the thickness of the epithelium and intact virions only apparent in the upper layers.
Infection with HPV is associated with an array of clinical conditions ranging from benign hyperplasias, such as warts and papillomas, to cancer, such as cervical, esophageal, oropharyngeal, vulval, vaginal, penile, and anal types. To date, more than 200 different HPV types have been isolated and are generally categorized into two groups (i.e., low-risk and high-risk) according to the frequency with which they are found in human cancer. Only a small fraction of those individuals infected with high-risk HPV actually develop cancer, which often occurs many years after the initial infection. HPV types 6 and 11 are low-risk types and are generally associated with over 90% of benign lesions. HPV-16 is the most common HPV type found associated with a malignant phenotype and together with HPV-18 exemplifies high-risk HPV types. About 30 HPV types have been identified that are spread through sexual contact and infect primarily the cervix, vagina, penis, and anus (anogenital regions). Of these types, there are four that are mostly found in malignant cells of cervical cancer: HPV-16, HPV-18, HPV31, and HPV-45. Together, HPV-16 and HPV-18 are responsible for 70% of cervical cancer. Worldwide, HPV has been implicated as an etiologic agent in 99.7% of cervical squamous cancer, with evidence that it also plays a role in adenocarcinomas of the cervix.
Many studies over the years, both epidemiologic and molecular in nature, have revealed the causative role of high-risk HPV types in human cancer, mostly with cervical cancer serving as the model system. From a public health viewpoint, once a causative role has been established, the diseases are largely preventable with the use of prophylactic anti-HPV vaccines. For cervical cancer, two such vaccines are available: GARDASIL®, which is directed against HPV types 6, 11, 16, and 18, and CERVARIX®, which is directed against HPV types 16 and 18 (Bosch F X, Castellsague X, de Sanjose S., Br. J. Cancer (2008), 98(1):15-21). However, it must be remembered that HPV infection alone is not sufficient for carcinogenesis. Indeed, additional oncogenic events within the host cell must occur in order for malignant transformation to result.
It is estimated that HPV is associated with 500,000 new cases of cervical cancer and 250,000 cervical cancer deaths worldwide each year. Within the US, it was estimated for 2008 that 11,070 new cases would be diagnosed, and about 3,870 women would die of their disease (Jemal A, Siegel R, Ward E, et al., CA Cancer J. Clin. (2008), 58(2):71-96). The disease usually presents in several premalignant stages ranging from mild dysplasia (cervical intraepithelial neoplasia grade 1 [CIN1]) to more severe degrees of neoplasia and microinvasive lesions (CIN2 or CIN3), to invasive cancer. Classification of the disease according to this CIN System forms the basis of diagnosis and treatment approaches including therapeutic options and secondary preventive measures. Importantly, CIN1 lesions can regress spontaneously with the risk of progression to severe dysplasia being 1% per year. Risk of progression of moderate dysplastic lesions is 16% within 2 years and 25% within 5 years.
Historically, the primary screening program for this disease has relied upon the cytologic appearance of abnormal cells in the transformation zone of the cervix (Pap test). The results of Pap tests are currently being reported according to the Bethesda classification system (revised in 2001), as outlined in Table 1. The comparable CIN histologic classification is also given.
TABLE 1Bethesda Classification System for Cervical Squamous Cell DysplasiaBethsesda SystemCIN SystemInterpretationNIL: Negative for intraepithelialNormalNo abnormal cellslesions or malignancyASC-US: Atypical squamous cells ofSquamous cells with abnormalities greaterundetermined significancethan those attributed to reactive changesASC-H: Atypical squamous cells,but do not meet the criteria for a squamouscannot exclude HSILintraepithelial lesionLSIL: Low-grade squamousCIN 1Mildly abnormal squamous cellsintraepithelial lesionsHSIL: High grade intraepithelialCIN 2/3Moderately or severely abnormal squamouslesions with features suspicious forcellsinvasions if suspectedCarcinomaInvasive squamous cellThe possibility of cancer is high enough tocarcinoma, invasive glandularwarrant immediate evaluation but does notcell adenocarcinomamean that the patient has cancer.
In the United States, about 55 million Pap smears are performed each year, and of these approximately 5% (2,750,000 smears per year) are diagnosed as containing atypical squamous cells of undetermined significance (ASC-US). It is known that about 39% of women with high grade disease (CIN2/CIN3 or frank cancer) will actually present as ASC-US. Thus, considering the 2,750,000 smears diagnosed as ASC-US each year, just under 10% have underlying CIN3 or cancer. The ASC-US and LSIL Triage Study (ALTS) was established as a NCI-sponsored multicenter randomized trial that would evaluate management options for this group of women including immediate colposcopy (costly), HPV DNA testing for high risk HPV types (less costly, using the Hybrid Capture 2 sold by Qiagen), and repeat cytology examinations (two follow-up visits) (“Human papillomavirus testing for triage of women with cytologic evidence of low-grade squamous intraepithelial lesions: baseline data from a randomized trial. The Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesions Triage Study (ALTS) Group. J. Natl. Cancer Inst. (2000), 92(5):397-402; and Solomon D, Schiffman M, Tarone R., J. Natl. Cancer Inst. (2001), 93(4):293-9). While the ALTS trial indicated that two repeat cytology examinations were as sensitive as HPV DNA testing for detecting CIN3 and cancer (CIN3+) in triaging women diagnosed with ASC-US for follow-up colposcopy, if only one repeat cytologic examination was performed due to cost, then this management option lost sensitivity. Thus, HPV DNA testing is considered an economically viable triaging strategy that should only be used in conjunction with the Pap test. In the study, it reduced the number of patients undergoing colposcopy by 44%. Patients over 21 with ASC-US who are positive for high-risk HPV DNA then, are referred for colposcopy and those who are negative are conservatively managed with repeat Pap smears at 6 and 12 months. In the ALTS trial it was found that of those referred to colposcopy based on a positive HPV test, about 28% actually had CIN2/3+, 23% had CIN1, and the remaining were normal upon biopsy. Clinical management then of this large group with equivocal or mildly abnormal tests every year where follow-up colposcopy is indicated upon a positive HPV test is daunting, and additional triage such as the introduction of the testing of new biomarkers would be desirable.
In patients with LSIL, it was found that 83% had positive HPV DNA tests with little predictive power of regression versus progression. According to the current NCCN Clinical Practice Guidelines in Oncology for Cervical Cancer Screening, women over 21 with ASC, LSIL, or HSIL are currently recommended to undergo colposcopic examination and subsequent treatment according to the biopsy histology. At this stage of patient management, the success of screening heavily depends on the accuracy of the colposcopically-directed biopsies of the approximately 2,000,000 women each year in the US who undergo colposcopy. Subsequent NCI-funded studies are directed both at improving biopsy procedures and the identification of biomarkers to assist in the diagnostic classification of patients who require additional treatment. Patients with normal or CIN1 results may be followed with repeat Pap tests or HPV DNA testing. For those with CIN2/3, further treatment is indicated such as loop electrosurgical excision procedure (LEEP), cryotherapy, cold-knife conization (CKC), or laser ablation. In this case, CIN2 patients are treated as a safety net since biomarkers to identify those lesions that will regress versus progress have as yet to be defined and fully evaluated. Total hysterectomy may be considered for women with CIN3. Diagnosis with invasive carcinoma requires total hysterectomy or external beam high-energy radiotherapy and implants with 192Ir, depending on the stage. Selected patients also benefit from chemotherapy.
Thus, despite the remarkable successes of cervical cancer screening programs in industrialized countries, improvement in the current screening procedures are warranted to identify women (young and old) with either high-grade disease or low-grade disease with the likelihood of progression, who will require follow-up and treatment for the disease, from those who will be submitted to unnecessary and costly medical procedures. This need arises not only from limitations within current screening tests such as inadequate sampling errors and false-positive and -negative rates, but also due to the inherent nature of the disease, in that as yet few, if any, robust biomarkers to risk-stratify low grade lesions into those likely to regress versus those likely to progress have been identified. Overall then, additional biomarkers of the disease are required, for which assays can be incorporated into screening programs to improve the sensitivity of early cervical cancer detection. From a global health issue, reduction in the costs of any such screening program clearly would positively impact screening coverage of such programs in low resource countries where cervical cancer is prevalent and death rates from the disease are higher.
Another cancer causally associated with HPV is anal cancer, though due to more limited data the exact percent caused by HPV and the most involved types of HPV have yet to be confirmed (Daling J R, Madeleine M M, Johnson L G, et al., Cancer (2004), 101(2):270-80). In the U.S. in 2008, 5,070 new cases are estimated to be diagnosed with 680 deaths. So while it is relatively uncommon in the general population, it occurs at a higher rate amongst at-risk populations such as those engaged in receptive anal intercourse. In men who have sex with men (MSM), approximately 40 per 100,000 are affected, with an even higher rate evident in HIV-infected MSM (Chin-Hong P V, Palefsky J M., Clin. Infect. Dis. (2002), 35(9):1127-34). Presumably the latter risk factor is due to a compromised immune response and enhanced failure to clear high risk HPV infections. One significant difference between anal HPV in MSM and cervical HPV infection in women is the age at which HPV is prevalent and at which cellular abnormalities are detected (Chin-Hong P V, Vittinghoff E, Cranston R D, et al., J. Natl. Cancer Inst. (2005), 97(12):896-905; and Franceschi S, Herrero R, Clifford G M, et al., Int. J. Cancer (2006), 119(11):2677-84). Cervical HPV infection is highly prevalent in the few years following initiation of sexual contact, but declines as women enter their 30's and 40's. In contrast, HPV prevalence and cytologic premalignant lesions of the anus in MSM appear to be higher in all adult age groups, an important consideration in the development of a screening program for anal cancer prevention in populations at risk.
As for cervical cancer, classification of premalignant lesions into anal intraepithelial neoplasia grades (AIN1-3) depends on the extent of dysplasia and evidence for neoplasia. The sensitivity for anal cytology for the detection of premalignant and malignant lesions is only around 70% in a high risk population, comparable to that for cervical cancer (Palefsky J M, Holly E A, Hogeboom C J, Berry J M, Jay N, Darragh T M., J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. (1997), 14(5):415-22). However, unlike cervical cancer, carcinogenic HPV testing as a screening tool may not be effective for several reasons: high prevalence of high risk HPV in at risk populations, more limited number of HPV types involved, and cross-reactivity of HPV testing with noncarcinogenic HPV types. Thus, for anal cancer in high risk populations, an effective screening program has yet to be defined to guide clinical practice. Currently, clinicians perform anoscopy or high-resolution anoscopy (HRA) as additional work-up, which in comparison to colposcopy may only be about 70% sensitive for the detection of premalignant lesions. While there is no consensus that a screening program is warranted for this disease, the introduction of an assay that reduces the number of MSM who undergo the relatively invasive and costly anoscopy procedure will potentially be cost-effective and greatly increase the efficiency with which those whom require immediate treatment are identified.
Over the past 15 years, despite a reduction in the number of people smoking cigarettes, the incidence of head and neck squamous cell cancer (HNSCC) has not declined. The growing subset of HNSCCs associated with HPV are thought to be largely responsible for this. The HPV-associated HNSCCs are commonly oropharyngeal and to a lesser extent pharyngeal in origin. Oropharyngeal cancers form in tissues of the oropharynx, which is the middle part of the throat and includes the soft palate, the base of the tongue, and the tonsils. These cancers seen in a younger non-smoking population are biologically and clinically distinct from carcinogen-induced HNSCC, often associated with sexual behavior, and overall have a better prognosis (Allen C T, Lewis J S, El-Mofty S K, Haughey B H, Nussenbaum B., Laryngoscope (2010), 120(9):1756-72, “Human Papillomaviruses and Cancer: Questions and Answers,” National Cancer Institute Fact Sheet, Pub. No. F703, revised Feb. 14, 2008). Early detection screening programs amongst at-risk populations are not in place, and often diagnosis is delayed.
Thus, there is a need for the early detection of HPV-associated precancers and cancers in patients so that appropriate treatment programs can be initiated early in the disease progression. It is an object of the present invention to provide methods for the early detection of HPV-associated precancers and cancers.